Defining the regenerative capacity of ductular cells from non-transplantable human liver

Liver disease killed 16,087 people in the UK in 2008, and causes 1 in 50 deaths in Scotland. Although a liver transplant is curative for these patients, a global shortage of donor organs means that many patients die waiting for suitable liver. Development of renewable therapeutic alternatives to whole organ transplants is imperative to alleviate this clinical shortage. Because of this clinical need a cellular treatment strategy would be highly attractive. Hepatocyte transplantation has successfully been used to: help keep patients alive who are waiting for whole liver transplantation; support pediatric patients in acute liver failure and correct metabolic liver disease. However, hepatocytes are not readily obtained and are usually sourced from discarded livers not suitable for transplantation. These livers are either significantly fatty or have undergone prolonged ischaemia (time without blood flow). These liver's cells are damaged and therefore high quality hepatocytes are difficult to obtain from such discarded livers. Furthermore it is not possible to expand the numbers of hepatocytes in the laboratory. An alternative source of transplantable cells for either biliary or hepatocyte regeneration would be a significant healthcare advance.

We have previously shown in the mouse that cells in liver called ductal cells or hepatic progenitor cells (HPCs) can act like stem cells and regenerate bile ducts and hepatocytes when the liver is severely damaged. Ductal cells/HPCs can be readily frozen and thawed without damage and then grown in the laboratory increasing the numbers of cells. We now aim to develop human ductal cells/HPCs for cell therapy with the eventual aim of developing a new type of cell therapy for liver patients. We aim to isolate these cells from livers that are too damaged to transplant whole.

Potential donor organs are increasingly undergoing normothermic perfusion prior to potential whole organ transplantation. For those livers deemed unsuitable for transplantation or where for logistical reasons the transplantation cannot proceed we will move seemlessly to efficient cell isolation of biliary ductal cells. Putative bipotential HPCs will be purified using the clinical cell sorter. These cells can be expanded and frozen/defrosted prior to use as a clinical cell therapy.

Over the course of the grant (48 months) we will:
1. Optimise the isolation and cell culture of ductular cells/HPCs from non-transplantable human livers.
2. Test the ability of HPCs to regenerate bile ducts +/- hepatocytes in mouse models. We need to know if the human cells are bipotential (regenerate bile ducts and hepatocytes) like the mouse ductular/HPC cells we described or are limited to bile duct regeneration alone. This result would guide future potential clinical conditions that the cells could be used for.
3. Develop protocols to isolate, purify and expand ductular/HPCs in a GMP compatible (suitable for use in man) manner as a potential clinical cell product.
4. Assess long term stability and safety of the transplanted ductular/HPCs.
5. If the above components are successful we will apply for MHRA (Medicines and Healthcare products Regulatory Agency) approval. This would pave the way for a fist in human clinical trial of ductular/HPC cell therapy. The above results would guide the clinical target to conditions requiring biliary or hepatocyte regeneration.

Hepatocyte transplantation has successfully been used to: bridge patients to whole liver transplantation; support paediatric patients in acute liver failure and correct metabolic liver disease. However, hepatocytes are usually sourced from discarded livers not suitable for transplantation that are significantly steatotic, meaning high quality hepatocytes are rarely obtained from these discarded livers. These same steatotic livers have abundant ductular reactions. Ductular reactions are activated in chronic liver disease and are thought to contain putative hepatic progenitor cells (HPCs), with the potential to regenerate hepatocytes and cholangiocytes. We have shown that mouse HPCs can: be frozen/defrosted without harm, be cultured and expanded in vitro without change of phenotype, repopulate and regenerate liver upon transplantation in a mouse model of induced hepatocyte injury and senescence, differentiate in vivo into hepatocytes and cholangiocytes.

We aim to develop human ductular cells/HPCs for liver disease therapy by:
1. refining our methods for isolating ductal cells/HPCs from human cadaveric livers discarded from transplantation using ex-situ normothermic perfusion prior and during cell isolation. HPCs will be purified using defined FACS protocols and their phenotype, gene expression profiles, clonogenicity, and phenotypic stability will be assessed.
2. to assess ductular cell/HPC biliary and hepatocyte regeneration potential, we will transplant defined populations of human HPCs into our immunodeficient mouse models of induced (i) hepatocyte or (ii) biliary senescence and injury, and assess histological and functional repopulation capacity.
4. Develop protocols to isolate, purify and expand ductular/HPCs in a GMP compatible manner as a potential clinical cell product.
5. Assess long term stability and safety of the transplanted ductular/HPCs.
6. If the above components are successful we will apply for MHRA approval to use the cells as an ATMP.

Liver disease is the 5th commonest cause of death in the UK and the deaths from cirrhosis are rapidly rising (British Liver Trust, www.britishlivertrust.org.uk ). 16,087 people in the UK died from liver disease in 20082, a 4.5% increase since 2007. In England the number of people who died from liver disease rose from 9,231 in 2001 to 11,575 in 2009 (Office of National Statistics, www.statistics.gov.uk). Most liver deaths were in people under 70, while one in 10 deaths of all people in their 40s were from liver conditions. Deaths from CLD now account for 1 in 50 of all Scottish deaths (Health in Scotland 2007: Annual Report of the Chief Medical Officer). These rising rates also contrast with falling mortality rates in recent years from the top 3 causes of mortality in Scotland: Coronary Heart Disease, Cerebrovascular Disease and Cancer (Overview of Scotland's Health and NHS Performance Audit Scotland 2007 http://www.audit-scotland.gov.uk/docs/health/2007/nr_071214_nhs_overview.pdf ).

The only curative option for end-stage liver disease is liver transplantation but donor organ availability cannot meet current demand and many patients die whilst waiting for a suitable organ. Typically, there are over 400 adults awaiting a liver transplant around the UK. Unfortunately due to the limited supply of donor organs a significant proportion of these patients will die whilst waiting for a liver transplantation (http://www.organdonation.nhs.uk/statistics). Furthermore many patients with severe liver disease are not eligible for transplantation.

If this study was successful and passed the milestones described then we would be in a position to then perform a phase 1 study (this would be the subject of a future funding application) with the input of the Cell Therapy CATAPULT (https://ct.catapult.org.uk/), who's aims include "Taking products into clinical trial, de-risking them for further investment". The exact clinical target is NOT defined here, but would be defined by the END of this study which will define whether ductular cells/HPCs are bipotential and can regenerate hepatocytes and biliary epithelia or are restricted to biliary epithelia alone.

The MRC Centre for Regenerative Medicine has a strong outreach program which will help disseminate results to appropriate patient groups and stakeholders.
Campbell has a track record of taking first in human cell therapies to the clinic.
Forbes and Oniscu are transplant Hepatologist and Surgeon respectively and perform clinical trials in this patient group. The Clinical Research facility, Little France has phase 1/first in human experience to deliver this potential cell therapy product.

Any intellectual property arising during this program would be appropriately protected with the input of the University and partners (Scottish National Blood Transfusion). Furthermore, the University of Edinburgh has a strong record of exploiting innovative research findings through commercialization of intellectual property (led through "Sunergos" our tech transfer company). To maximize the scientific and clinical impact from this regenerative medicine the investigators will closely involve the regenerative network provided by the UKRMP hubs of which Forbes is a director of the Niche Hub. The investigators have a record of publishing in high impact journals with wide readership in the relevant scientific and clinical communities. The MRC Centre for Regenerative Medicine has a strong outreach program which will help disseminate results to appropriate patient groups and stakeholders.